World Journal of GastroenterologyWorld J Gastroenterol 2017 December 7; 23(45): 7945-8108

ISSN 1007-9327 (print)ISSN 2219-2840 (online)

Published by Baishideng Publishing Group Inc

S

MINIREVIEWS7945 Pancreaticacinarcellcarcinoma:Areviewonmolecularprofilingofpatienttumors

Al-Hader A, Al-Rohil RN, Han H, Von Hoff D

7952 Probioticsforgastrointestinaldisorders:ProposedrecommendationsforchildrenoftheAsia-Pacificregion

Cameron D, Hock QS, Kadim M, Mohan N, Ryoo E, Sandhu B, Yamashiro Y, Jie C, Hoekstra H, Guarino A

ORIGINAL ARTICLEBasic Study

7965 Down-regulationofmiR-30a-3p/5ppromotesesophagealsquamouscellcarcinomacellproliferationby

activatingtheWntsignalingpathway

Qi B, Wang Y, Chen ZJ, Li XN, Qi Y, Yang Y, Cui GH, Guo HZ, Li WH, Zhao S

7978 MesenchymalstemcellsrescueacutehepaticfailurebypolarizingM2macrophages

Li YW, Zhang C, Sheng QJ, Bai H, Ding Y, Dou XG

7989 Improvedexperimentalmodelofhepaticcystichydatiddiseaseresemblingnaturalinfectionroutewith

stablegrowingdynamicsandimmunereaction

Zhang RQ, Chen XH, Wen H

Case Control Study

8000 Recurrenceinnode-negativeadvancedgastriccancer:Novelfindingsfromanin-depthpathological

analysisofprognosticfactorsfromamulticentricseries

Baiocchi GL, Molfino S, Baronchelli C, Giacopuzzi S, Marrelli D, Morgagni P, Bencivenga M, Saragoni L, Vindigni C,

Portolani N, Botticini M, De Manzoni G

Retrospective Cohort Study

8008 Albuminasaprognosticmarkerforulcerativecolitis

Khan N, Patel D, Shah Y, Trivedi C, Yang YX

8017 Pretransplantationfetal-maternalmicrochimerisminpediatriclivertransplantationfrommother

Yi NJ, Park MS, Song EY, Ahn HY, Byun J, Kim H, Hong SK, Yoon K, Kim HS, Ahn SW, Lee HW, Choi Y, Lee KW, Suh KS,

Park MH

Retrospective Study

8027 Impactofhomogeneouspathologicresponsetopreoperativechemotherapyinpatientswithmultiple

colorectallivermetastases

Sabbagh C, Chatelain D, Attencourt C, Joly JP, Chauffert B, Cosse C, Regimbeau JM

Contents Weekly Volume 23 Number 45 December 7, 2017

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ContentsWorld Journal of Gastroenterology

Volume 23 Number 45 December 7, 2017

8035 Two-stepmethodforcreatingagastrictubeduringlaparoscopic-thoracoscopicIvor-Lewisesophagectomy

Liu Y, Li JJ, Zu P, Liu HX, Yu ZW, Ren Y

Observational Study

8044 Clinicalvalueofliverandspleenshearwavevelocityinpredictingtheprognosisofpatientswithportal

hypertension

Zhang Y, Mao DF, Zhang MW, Fan XX

Prospective Study

8053 Genderdifferencesinghrelin,nociceptiongenes,psychologicalfactorsandqualityoflifeinfunctional

dyspepsia

Choi YJ, Park YS, Kim N, Kim YS, Lee SM, Lee DH, Jung HC

Randomized Clinical Trial

8062 Efficacyofcombinationtherapywithnatriureticandaquareticdrugsincirrhoticascitespatients:A

randomizedstudy

Uojima H, Hidaka H, Nakayama T, Sung JH, Ichita C, Tokoro S, Masuda S, Sasaki A, Koizumi K, Egashira H, Kako M

SYSTEMATIC REVIEW8073 Double-balloonenteroscopy-assisteddilatationavoidssurgeryforsmallbowelstrictures:Asystematicreview

Baars JE, Theyventhiran R, Aepli P, Saxena P, Kaffes AJ

META-ANALYSIS8082 Impactofinflammatoryboweldiseaseactivityandthiopurinetherapyonbirthweight:Ameta-analysis

Gonzalez-Suarez B, Sengupta S, Moss AC

CASE REPORT8090 Midgutneuroendocrinetumorpresentingwithacuteintestinalischemia

Mantzoros I, Savvala NA, Ioannidis O, Parpoudi S, Loutzidou L, Kyriakidou D, Cheva A, Intzos V, Tsalis K

8097 Endoscopicsubmucosaldissectioninapatientwithesophagealadenoidcysticcarcinoma

Yoshikawa K, Kinoshita A, Hirose Y, Shibata K, Akasu T, Hagiwara N, Yokota T, Imai N, Iwaku A, Kobayashi G, Kobayashi H,

Fushiya N, Kijima H, Koike K, Kanayama H, Ikeda K, Saruta M

8104 Simultaneousliver,pancreas-duodenumandkidneytransplantationinapatientwithhepatitisBcirrhosis,

uremiaandinsulindependentdiabetesmellitus

Li J, Guo QJ, Cai JZ, Pan C, Shen ZY, Jiang WT

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NAMEOFJOURNALWorld Journal of Gastroenterology

ISSNISSN 1007-9327 (print)ISSN 2219-2840 (online)

LAUNCHDATEOctober 1, 1995

FREQUENCYWeekly

EDITORS-IN-CHIEFDamian Garcia-Olmo, MD, PhD, Doctor, Profes-sor, Surgeon, Department of Surgery, Universidad Autonoma de Madrid; Department of General Sur-gery, Fundacion Jimenez Diaz University Hospital, Madrid 28040, Spain

Stephen C Strom, PhD, Professor, Department of Laboratory Medicine, Division of Pathology, Karo-linska Institutet, Stockholm 141-86, Sweden

Andrzej S Tarnawski, MD, PhD, DSc (Med), Professor of Medicine, Chief Gastroenterology, VA Long Beach Health Care System, University of Cali-fornia, Irvine, CA, 5901 E. Seventh Str., Long Beach,

CA 90822, United States

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Contents

EDITORS FOR THIS ISSUE

Responsible Assistant Editor: Xiang Li Responsible Science Editor: Ke ChenResponsible Electronic Editor: Yan Huang Proofing Editorial Office Director: Jin-Lei WangProofing Editor-in-Chief: Lian-Sheng Ma

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World Journal of GastroenterologyVolume 23 Number 45 December 7, 2017

Editorial boardmember ofWorld Journal ofGastroenterology ,Marc EMartignoni,MD,AssociateProfessor,DepartmentofGeneralSurgery,TechnischeUniversitätMünchen,Munic81675,Germany

World Journal of Gastroenterology (World J Gastroenterol, WJG, print ISSN 1007-9327, online ISSN 2219-2840, DOI: 10.3748) is a peer-reviewed open access journal. WJG was estab-lished on October 1, 1995. It is published weekly on the 7th, 14th, 21st, and 28th each month. The WJG Editorial Board consists of 1375 experts in gastroenterology and hepatology from 68 countries. The primary task of WJG is to rapidly publish high-quality original articles, reviews, and commentaries in the fields of gastroenterology, hepatology, gastrointestinal endos-copy, gastrointestinal surgery, hepatobiliary surgery, gastrointestinal oncology, gastroin-testinal radiation oncology, gastrointestinal imaging, gastrointestinal interventional ther-apy, gastrointestinal infectious diseases, gastrointestinal pharmacology, gastrointestinal pathophysiology, gastrointestinal pathology, evidence-based medicine in gastroenterol-ogy, pancreatology, gastrointestinal laboratory medicine, gastrointestinal molecular biol-ogy, gastrointestinal immunology, gastrointestinal microbiology, gastrointestinal genetics, gastrointestinal translational medicine, gastrointestinal diagnostics, and gastrointestinal therapeutics. WJG is dedicated to become an influential and prestigious journal in gas-troenterology and hepatology, to promote the development of above disciplines, and to improve the diagnostic and therapeutic skill and expertise of clinicians.

World Journal of Gastroenterology (WJG) is now indexed in Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®), Journal Citation Reports®, Index Medicus, MEDLINE, PubMed, PubMed Central and Directory of Open Access Journals. The 2017 edition of Journal Citation Reports® cites the 2016 impact factor for WJG as 3.365 (5-year impact factor: 3.176), ranking WJG as 29th among 79 journals in gastroenterology and hepatol-ogy (quartile in category Q2).

I-IX EditorialBoard

ABOUT COVER

INDEXING/ABSTRACTING

AIMS AND SCOPE

FLYLEAF

��� December 7, 2017|Volume 23|�ssue 45|WJG|www.wjgnet.com

Yan-Wei Li, Chong Zhang, Qiu-Ju Sheng, Han Bai, Yang Ding, Xiao-Guang Dou, Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang 110022, Liaoning Province, China

ORCID number: Yan-Wei Li (0000-0001-6679-7460); Chong Zhang (0000-0002-9434-786X); Qiu-Ju Sheng (0000-0001-7561 -7396); Han Bai (0000-0001-7228-8689); Yang Ding (0000-0003 -0836-7790); Xiao-Guang Dou (0000-0002-5293-6009).

Author contributions: Li YW, Zhang C, Sheng QJ, Bai H, Ding Y, and Dou XG substantially contributed to the conception and design of the study and acquisition, analysis, and interpretation of the data; all authors drafted the article, made critical revisions related to the intellectual content of the manuscript, and approved the final version of the article to be published.

Supported by Liaoning Provincial Science and Technology Key Project for Translational Medicine, No. 2014225020; Outstanding Scientific Fund of Shengjing Hospital, No. 201102; and Liaoning Provincial Science and Technology Key Project for Translational Medicine, No. 2016509.

Institutional review board statement: The study was reviewed and approved by Shengjing Hospital of China Medical University Institutional Review Board.

Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Ethics Committee of Shengjing Hospital of China Medical University Institutional. (IACUC protocol number: 2016PS248K).

Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.

Data sharing statement: The technical appendix, statistical code and dataset are available from the first author Yan-Wei Li at liyanwei201510296@163.com. All the participants gave informed consent for data sharing.

Open-Access: This article is an open-access article which was

selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

Manuscript source: Unsolicited manuscript

Correspondence to: Xiao-Guang Dou, PhD, Chief Doctor, Department of Infectious Diseases, Shengjing Hospital of China Medical University, NO.39 Huaxiang Road, Shenyang 110022, Liaoning Province, China. douxg@sj-hospital.orgTelephone: +86-24-9661562211Fax: +86-24-25998744

Received: August 31, 2017Peer-review started: September 3, 2017First decision: September 20, 2017Revised: October 1, 2017Accepted: November 1, 2017Article in press: November 1, 2017Published online: December 7, 2017

AbstractAIMTo investigate whether M1 or M2 polarization contri-butes to the therapeutic effects of mesenchymal stem cells (MSCs) in acute hepatic failure (AHF).

METHODSMSCs were transfused into rats with AHF induced by D-galactosamine (DGalN). The therapeutic effects of MSCs were evaluated based on survival rate and hepatocyte proliferation and apoptosis. Hepatocyte regeneration capacity was evaluated by the expression

7978 December 7, 2017|Volume 23|Issue 45|WJG|www.wjgnet.com

ORIGINAL ARTICLE

Mesenchymal stem cells rescue acute hepatic failure by polarizing M2 macrophages

Basic Study

Yan-Wei Li, Chong Zhang, Qiu-Ju Sheng, Han Bai, Yang Ding, Xiao-Guang Dou

Submit a Manuscript: http://www.f6publishing.com

DOI: 10.3748/wjg.v23.i45.7978

World J Gastroenterol 2017 December 7; 23(45): 7978-7988

ISSN 1007-9327 (print) ISSN 2219-2840 (online)

of the hepatic progenitor surface marker epithelial cell adhesion molecule (EpCAM). Macrophage polarization was analyzed by M1 markers [CD68, tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), inducible nitric oxide synthase (INOS)] and M2 markers [CD163, interleukin (IL)-4, IL-10, arginase-1 (Arg-1)] in the survival and death groups after MSC transplantation.

RESULTSThe survival rate in the MSC-treated group was in-creased compared with the DPBS-treated control group (37.5% vs 10%). MSC treatment protected rats with AHF by reducing apoptotic hepatocytes and promoting hepatocyte regeneration. Immunohistochemical analysis showed that MSC treatment significantly increased the expression of EpCAM compared with the control groups (P < 0.001). Expression of EpCAM in the survival group was significantly up-regulated compared with the death group after MSC transplantation (P = 0.003). Trans-plantation of MSCs significantly improved the expression of CD163 and increased the gene expression of IL-10 and Arg-1 in the survival group. IL-4 concentrations were significantly increased compared to the death group after MSC transplantation (88.51 ± 24.51 pg/mL vs 34.61 ± 6.6 pg/mL, P < 0.001). In contrast, macrophages showed strong expression of CD68, TNF-α, and INOS in the death group. The concentration of IFN-γ was significantly increased compared to the survival group after MSC transplantation (542.11 ± 51.59 pg/mL vs 104.07 ± 42.80 pg/mL, P < 0.001).

CONCLUSIONM2 polarization contributes to the therapeutic effects of MSCs in AHF by altering levels of anti-inflammatory and pro-inflammatory factors.

Key words: Acute hepatic failure; Mesenchymal stem cells; Macrophages; Polarization; Inflammation

© The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.

Core tip: M1 or M2 polarization governs the therapeutic effect of acute hepatic failure (AHF). Mesenchymal stem cells (MSCs) were transfused into rats with AHF induced by galactosamine. It was found that MSCs alleviated the survival rate and biochemical indicators by promoting hepatocyte regeneration. Immunohistochemistry, flow cytometry, and RT-PCR showed that M2 polarization contributes to the rescue of AHF by MSCs in the survival group after MSC transplantation. In addition, in the death group after MSC transplantation, the number of M1 macrophages increased significantly. Our findings suggest that M2 polarization contributes to the rescue of AHF by MSCs, which result in altered levels of anti-inflammatory and pro-inflammatory factors.

Li YW, Zhang C, Sheng QJ, Bai H, Ding Y, Dou XG. Mesenchymal stem cells rescue acute hepatic failure by

polarizing M2 macrophages. World J Gastroenterol 2017; 23(45): 7978-7988 Available from: URL: http://www.wjgnet.com/1007-9327/full/v23/i45/7978.htm DOI: http://dx.doi.org/10.3748/wjg.v23.i45.7978

INTRODUCTIONAcute hepatic failure (AHF) is a lethal condition ch­aracterized by widespread hepatocyte necrosis, acute deterioration of liver function, and subseq­uent multiorgan failure. Although many animal and preliminary human studies have shown that stem cell transplantation has substantial potential in treating AHF[1­4], transplantation has rarely produced satisfying therapeutic effects. The exact mechanism through which stem cells assist in organ repair remains elusive. Recent studies have also indicated a substantial role for paracrine effects in delivering overall benefits, although specific cells and signaling molecules have not been identified to mediate these paracrine effects[5]. However, macrophages in the liver play an indispensable role in paracrine mechanisms. There has been a major paradigm shift in the field of macrophage biology with the recognition that macrophages play an important role in homeostasis[6]. Several studies employing selective Kupffer cell depletion in rodents have explored the role of this cell type in hepatocyte proliferation and liver regeneration following partial hepatectomy[7,8]. However, there is little information available regarding the role of macrophages in hepatocyte proliferation. This may be due to the multi­phenotype and multi­functional roles of macrophages in liver regeneration, as they are a major source of both pro­proliferative and anti­proliferative mediators in the liver. Classically activated macrophages (M1 macrophages) mediate host defenses from a variety of bacteria, protozoa, and viruses and have roles in anti­tumor immunity. M2 macrophages have an anti­inflammatory function and regulate wound healing[9,10]. Different phenotypes play various roles in tissue damage and maintenance[11­17]. For example, M1 macrophages are induced by exposure to CD68[18] and are associated with the phagosomes of macrophages, which is consistent with enhanced phagocytosis. These macrophages are characterized by the expression of high levels of inducible nitric oxide synthase (INOS) induced by interferon­γ (IFN­γ) that liberates pro­inflammatory cytokines, such as tumor necrosis factor alpha (TNF­α) and interleukin (IL)­6, which are increased in inflammatory reactions and tissue injury. In contrast, M2 macrophages are characterized by exposure to CD163[19], which is expressed by liver Kupffer cells. These macrophages are activated by IL­4, express high levels of arginase­1 (Arg­1), and release immune­modulatory mediators (such as IL­10) to modulate the inflammatory response and to promote

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Li YW et al . M2 polarization in MSC transplantation

tissue remodeling. MSC transplantation may be useful in treating AHF

conditions. In the present study, we evaluated the contribution of M1 and M2 macrophages in survival and death groups after MSC transplantation to investigate whether macrophage polarization contributes to the rescue of AHF by MSCs.

MATERIALS AND METHODSAHF animal model Male Wistar rats weighing 190 ± 20 g were obtained from the Huafukang Experimental Animal Center (Beijing, China). The study was reviewed and approved by the Ethics Committee of Shengjing Hospital of China Medical University. The animal study protocol, in compliance with the Guidelines of China for Animal Care, conformed to internationally accepted principles in the care and use of experimental animals. Animals were housed at room temperature (22 ± 2 ℃) with light cycles between 08:00 and 22:00 and free access to food and water. A total of 52 rats were randomly divided into four groups: an experimental group (group A, n = 16), a control group (group B, n = 10), an MSC–treated group (group C, n = 16), and a DPBS (Dulbecco phosphate­buffered saline)–treated group (group D, n = 10). Rats in group A were injected intraperitoneally (i.p.) with D­galactosamine (DGalN) (1.2 g/kg; Sigma­Aldrich, St. Louis, MO, United States). Rats in group B were injected i.p. with 2 mL of 0.9% phosphate buffered saline (PBS). At 12 h after DGalN treatment, rats in group C underwent intravenous tail vein transplantation of 5.5 × 105 MSCs dissolved in 1.0 mL of DPBS, and rats in group D were given 1.0 mL of DPBS. All rats were selected for survival analysis at 72 h after treatment. The survival rate of rats remained unchanged at 48 h after treatment. The rats in the survival group were still in good physical condition at 48 h after MSC treatment. The rats in the death group were in poor physical condition or in the state of death before they died at 48 h after MSC treatment. Serum and liver tissues were collected at 48 h after MSCsGFP transplantation for biochemical analyses, inflammatory factor detection, and further evaluation.

MSCsGFP culture and MSCsGFP transplantationWistar bone marrow MSCs were obtained from a cell bank (Shanghai, China) and cultured in α­MEM medium with GlutaMAX­I (Gibco, United States), supplemented with 10% fetal bovine serum (Gibco, United States), 100 IU/mL penicillin and 100 μg/mL streptomycin (Thermo, United States). When cells reached 80%­90% confluence, they were trypsinized with 0.05 g/L trypsin-EDTA (Gibco, United States) and replated at a density of 1 × 10 4/cm2 for further expansion. After cells were passaged to the fourth generation, they were infe­cted with an adenovirus encoding the gene encoding green fluorescent protein (GFP), and the multiplicity

of infection was determined by fluorescence inverted phase­contrast microscopy.

Biochemical assay and histological evaluationSerum levels of alanine aminotransferase (ALT), as­partate aminotransferase (AST), albumin (ALB), and total bilirubin (TBIL) were monitored with an automatic analyzer (Roche, United States) and liver biochemical indicators were estimated. The liver was fixed in 4% paraformaldehyde for hematoxylin and eosin (HE) or immunohistochemical staining. Paraffin­embedded liver tissue was cut into 3­μm thick sections for his­topathological evaluation, deparaffinized in xylene, and rehydrated through a series of decreasing concen–trations of ethanol. Sections were stained with HE and analyzed under a light microscope.

Immunohistochemistry and immunofluorescence stainingImmunohistochemistry was performed with primary rabbit or mouse anti­rat antibodies (Abcam, Cambridge, MA, United States) for EpCAM, CD68, and CD163. Liver sections were deparaffinized in xylene and re­hydrated through a series of decreasing concentra­tions of ethanol. Heat­mediated antigen retrieval was performed using citrate buffer (MVS­0100, MXB Blotechnologies, Fujian, China). Blocking solution and secondary antibodies (KIT­9710, MXB Blotechnologies, Fujian, China) were applied according to standard protocols. Sections were incubated overnight with a primary antibody at 4 ℃ and visualized with DAB (ZLI­9017, ZSGB­BIO, Beijing, China).

Indirect immunofluorescence was used to detect the phenotype of M1/M2 following overnight incubation at 4 ℃ with primary antibodies. Secondary antibodies (Abcam, Cambridge, MA, United States) were used at room temperature for 4 h with goat anti­mouse IgG­H&L (Abcam, Cambridge, MA, United States) and goat anti­rabbit IgG­H&L (Abcam, Cambridge, MA, United States). Nuclear staining was performed using DAPI (ZSGB­BIO, Beijing, China). A standard in situ TUNEL (Roche, Indianapolis, IN, United States) method was used for detection of DNA fragmentation in apoptotic cells according to the manufacturer’s instructions. Cell proliferation was determined using anti­Ki67 (Novus, NB500­170).

To determine engraftment of MSCs after GFP transfection, the livers from rats in the survival and death groups were dissected out, fixed in 4% formaldehyde and optimal cutting temperature (OCT) compound (Tissue­Tek, Sakura, Japan), and preserved at ­20 ℃. GFP expression by transplanted MSCs was detected by fluorescence inverted phase-contrast microscopy.

Measurement of cytokine productionCytokine production was measured in serum centri­fuged at 1500 r/min for 15 minutes. IL­4 and IFN­γ were tested using Multi­Analyte Flow Assay Kit

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and 30% (3/10) of the animals had survived in the MSC­ and DPBS­treated groups, respectively. At 48 h after transplantation, survival in the MSC­ and DPBS­treated groups decreased to 37.5% (6/16) and 10% (1/10), respectively (Figure 1C). Although there was no statistical significance, survival rate was increased by MSC transplantation. At 24 h and 48 h after MSC transfusion, biochemical indicators (ALT/AST/ALB/TBIL) had significantly changed compared with rats in the DPBS­treated group (Figure 1). To investigate the liver histology of rats with AHF after MSC transplantation, HE staining was conducted (Figure 1). At 48 h after MSC transfusion, no obvious histopathological changes were observed in rats infused with MSCs, and most of the tissue showed generalized necrotic areas. Five days after transplantation, most of the tissue had returned to normal with only a few necrotic areas, indicating liver tissue repair after liver function repair.

MSC transfusion promotes hepatocyte regenerationTo determine whether MSC treatment promotes hepatocyte regeneration compared to rats in the DPBS­treated group, Ki67­positive hepatocytes were detected. Ki67­positive hepatocytes significantly increased at 48 h after transplantation (P < 0.001) (Figure 2). In DPBS­treated rats with AHF, many TUNEL­positive hepatocytes were observed, yet only a few hepatocytes were observed after MSC treatment (P < 0.001).

EpCAM has been shown to be expressed in a population of rat oval cells, which are composed of liver progenitors[16,17]. In the present study, significant up­regulation of EpCAM was observed in the MSC­treated group compared with DPBS­treated group (P < 0.001) (Figure 3). Compared with the death group, EpCAM expression was increased in the survival group after MSC transplantation (P = 0.003), suggesting the vital roles of progenitor cells in the regeneration process after MSC transplantation.

Enhanced M2 polarization in the survival group after MSC transplantationTo investigate the role of macrophage subsets in AHF, liver sections were stained for the recently described M1/M2 specific markers CD68 and CD163, which preferentially detect invading macrophages. The number of CD68+ macrophages was obviously up­regulated in the DGalN­treated group (Figure 4). However, compared to the death group after MSC transplantation, a significantly greater number of CD163+ macrophages was observed in the survival group, while the number of CD68+ macrophages was decreased (Figure 5). Serum protein levels of IL­4 were significantly higher in the survival group than in the death group (88.51 ± 24.51 pg/mL vs 34.61 ± 6.6 pg/mL, P < 0.001) (Figure 6). The mRNA expression of IL-10 and Arg-1 was significantly up-regulated in the survival group (P < 0.001) (Figure 7).

(BioLegend, CA, United States) and analyzed by flow cytometry. Each analysis was performed in duplicate. In this quantitative assay system, specific antibodies directed against each cytokine are conjugated to the surface of fluorescence­coded microbeads, with each fluorescence­coded microbead type being conjugated to one specific capture antibody.

Quantitative real-time PCRTotal RNA was extracted from liver tissue (~100 mg) using TRIzol reagent (Invitrogen, United States) according to the manufacturer’s instructions, and the amount of isolated RNA was estimated by ribogreen fluorescence. Purity was assessed by the absorbance ratio at 260 and 280 nm. A total of 3 μg was reverse­transcribed using a High Capacity cDNA Reverse Transcription Kit (Promega, United States). Real­time quantitative PCR was performed using SYBR Green I Master and the appropriate primers on a LightCycler 480 instrument. In parallel, mRNA levels of human housekeeping GAPDH were analyzed as an internal normalization control. Primers used are shown in Table 1. Data were calculated using the ΔCt method and were normalized to GAPDH.

Statistical analysisSurvival statistics were assessed using Log­rank (Mantel­Cox) test. Data are expressed as the mean ± SD. Differences between groups were analyzed by independent sample t-test. Serum concentra­tion and gene expression of cytokine assays were performed in duplicate or triplicate for each specific sample. All data points are the mean of duplicate or triplicate measurements. Differences were considered statistically significant at P < 0.05.

RESULTSSurvival rate is increased and biochemical indicators are altered by MSC transplantationImplanted MSCs were observed in the liver of treated rats (Figure 1). At 24 h after treatment, 50% (8/16)

Table 1 Primers used in mRNA expression analysis

Gene name Sequence

GAPDH (Forward) 5’-GGCACAGTCAAGGCTGAGAATG-3’(Reverse) 5’-ATGGTGGTGAAGACGCCAGTA-3’

CD68 (Forward) 5’-TCGGGCCATGCTTCTCTT-3’(Reverse) 5’-AGGGGCTGGTAGGTTGATTGT-3’

CD163 (Forward) 5’-CTGGGATGTCCAACTGCCAT-3’(Reverse) 5’-AATGCTTCCCCCATTCCTGG-3’

Arg-1 (Forward) 5’-GCTGTGGTAGCAGAGACCCAGA-3’(Reverse) 5’-CATCCACCCAAATGACGCATAG-3’

IL-10 (Forward) 5’-CAGACCCACATGCTCCGAGA-3’(Reverse) 5’-CAAGGCTTGGCAACCCAAGTA-3’

Nos2 (Forward) 5’-TCCTCAGGCTTGGGTCTTGTTAG-3’(Reverse) 5’-TTCAGGTCACCTTGGTAGGATTTG-3’

TNF-α (Forward) 5’-CCGATTTGCCACTTCATACCA-3’(Reverse) 5’-TAGGGCAAGGGCTCTTGATG-3’

Li YW et al . M2 polarization in MSC transplantation

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Enhanced M1 polarization in the death group after MSC transplantationIn the death group after MSC transplantation, the number of CD68+ macrophages was significantly increased and the number of CD163+ macrophages was markedly reduced. We investigated serum IFN­γ protein levels and showed that the concentration of IFN­γ was significantly up-regulated in the death group (542.11 ± 51.59 pg/mL vs 104.07 ± 42.80 pg/mL, P < 0.001) (Figure 6). TNF­α and INOS gene expression was dramatically increased (P < 0.001) (Figure 7).

DISCUSSIONAs a heterogeneous population of cells, MSCs have the potential for multilineage differentiation. MSCs can differentiate into a variety of liver cells under appropriate culture conditions[20­23]. Many clinical studies have indicated that MSCs are safe and effective in clinical studies and are useful to treat hepatic failure[24­26]. In the

present study, MSC infusion was beneficial in improving the survival rate and liver histopathology after altering the concentration of biochemical indicators. To study the reasons for increased survival in the MSC­treated group, we analyzed the expression of EpCAM, which is a marker used to assess liver regeneration[27,28]. The additional study showed that in the death group, implanted MSCs did not fully translate into functional liver cells after treatment and that subsequent liver cell hepatocyte proliferation was unsatisfactory, which cannot completely improve hepatocyte inflammatory necrosis.

There is growing evidence that MSCs increase angiogenesis and improve local cell function through paracrine effects, which are involved in releasing growth factors and signaling molecules[5,29­33]. The pivotal role of paracrine effects in stem cell therapies has been recognized to contribute to many biological processes, such as preventing inflammation, inhibiting apoptosis, improving metabolism, and promoting regeneration.

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Figure 1 Survival rate and biochemical indicators in rats are improved after mesenchymal stem cell treatment. Colonization of mesenchymal stem cells (MSCs) was observed in the liver (A and B). The red arrow on the left shows engraftment of MSCs and nuclear staining in the same slice. Survival rates were compared between the MSC-treated group and the DPBS-treated group at each time point (C) (P = 0.36). Serum samples collected at various times (0 h, 24 h, 48 h) after MSC treatment were analyzed for levels of ALT, AST, ALB, and TBIL and compared with the DPBS-treated group (D-G). HE staining of liver sections was performed in each group. Compared with the PBS-treated group (H), we observed necrosis of centrilobular hepatocytes, characterized by cell shrinkage and lost nuclei, interstitial hemorrhage, and inflammatory cell infiltration in the DPBS-treated group (I). Liver histomorphology at 48 h after MSC treatment (J) did not change significantly compared with the DPBS-treated group, but the number of hepatocytes with edema, shrinkage, and lost nuclei decreased significantly, with massive inflammatory cell infiltration and increased number of cells observed. The liver histomorphology was gradually repaired after 5 d (K).

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Macrophages are the major cells involved in paracrine effects. We found that M2 macrophages and their associated cytokines can contribute to AHF rescuing by MSCs. The number of CD163+ macrophages and levels of IL­10 and Arg­1 were significantly up­regulated in the survival group. In contrast, CD68+ macrophages and levels of TNF­α and INOS were significantly up­regulated in the death group. During type 2 helper T (TH2)­mediated immune responses, IL­4 can induce macrophages undergoing M2 activation[34], leading to expansion beyond a continuum in multiple activation states. In response to IFN­γ, macrophages undergo M1 activation during type 1 (TH1)­mediated immune responses and represent another extreme in terms of

activation states. Our study demonstrates that high IL­4 levels drive M2 polarization, which occurred in the survival group after MSC transplantation. High expression of IFN­γ in the death group stimulated macrophages to undergo M1 activation.

In this study, we investigated the role of macrophage polarization in AHF rescuing by MSCs and found that polarized macrophages from the M2 anti-inflammatory phenotype promote MSC activity. Macrophages to M2 polarization also increase infused MSC activity during myocardial and spinal cord injuries[35,36]. Tremendous research efforts have corroborated the concept that hepatic macrophages are central in the pathogenesis of acute hepatic injury. Elsegood et al[37] showed that

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Figure 2 Assessment of hepatocyte apoptosis and proliferation after mesenchymal stem cell transplantation. Immunofluorescence for Ki-67 (A and B) and terminal deoxyribonucleotide transferase (TdT)-mediated deoxyuridine triphosphate nick end labeling (TUNEL) (C and D) staining in MSC-treated and DPBS-treated livers. A and C: MSC-treated group; B and D: DPBS-treated group. The numbers of Ki-67-positive and TUNEL-positive hepatocytes were observed in the DPBS- and MSC-treated groups (E and F). Bar represents the mean ± SD. (n = 5, bP < 0.001). MSC: Mesenchymal stem cell.

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the number of macrophages increases in the liver to induce liver progenitor cell proliferation in chronic liver injury models. Our data suggest that the number of macrophages was increased in the pathogenesis of acute hepatic injury. Importantly, the number of M1 macrophages was increased significantly compared to M2 macrophages. Lanthier et al[11] reported that higher liver macrophage expansion could increase proliferative hepatocytes and is associated with a favorable outcome. Here, we determined that TNF­α expression depressed hepatocyte regeneration in AHF. These results differ from those of Lanthier et al[11] and Bihari et al[38], who reported that TNF­α levels contribute to liver cell proliferation in chronic hepatic injury. This

disparity could reflect differences in the mechanisms of hepatocyte repair in acute and chronic liver injury. Our results show an increase in IL­10 gene expression in the survival group. Interestingly, these results are in agreement with the suggestion that IL­10 released by MSCs has the potential for therapeutic recovery of liver fibrosis[39,40].

MSCs improve liver function, although the specific mechanism of action is still unknown. Several studies have shown that MSCs have immunomodulatory properties, focusing on their paracrine effect. Studies of macrophage functions in hepatocyte repair have typically not distinguished between M1 and M2 after MSC transplantation. EpCAM+ hepatocytes are able to

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Figure 3 Immunohistochemical staining for epithelial cell adhesion molecule expression in each group. A: Survival group after mesenchymal stem cell (MSC) treatment; B: Death group after MSC treatment; C: Survival group after DPBS treatment; D: Death group after DPBS treatment; E: Integrated optical density of immunohistochemical staining for EPCAM+ hepatocytes. Bar represents the mean ± SD (n = 5, aP < 0.05, bP < 0.001).

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differentiate into cholangiocytes or hepatocytes and are located in the portal area. CD68+ macrophages and CD163+ macrophages are mainly located in the portal

zone. However, a specific signaling pathway between macrophage polarization, associated cytokines, and hepatocyte regeneration has not been examined to

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Figure 4 Immunohistochemistry for polarization of macrophages in liver tissue. A: Distribution of macrophages reacting to CD68 for M1 in PBS-treated group; B: Distribution of macrophages reacting to CD68 for M1 in DGalN-treated group; C: Distribution of macrophages reacting to CD163 for M2 in PBS-treated group; D: Distribution of macrophages reacting to CD163 for M2 in DGalN-treated group (n = 4).

A B C D

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Figure 5 Immunofluorescence for polarization of macrophages in liver tissue. A-D: Death group after mesenchymal stem cell (MSC) treatment; E-H: Survival group after MSC treatment. Green fluorescence indicates CD163+ macrophages. Red fluorescence indicates CD68+ macrophages. Nuclei are stained blue with DAPI. Immunofluorescence for M1 macrophages reacting to CD68 and M2 macrophages reacting to CD163 is shown (n = 5).

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date.In conclusion, MSCs transfused into rats were

recruited and increased the survival rate by inhibiting apoptotic hepatocytes and promoting hepatocyte regeneration. This study demonstrates that expression of hepatic progenitor surface marker (EpCAM) is the key to improving the prognosis of AHF. Although this study lacks specific cell numbers of macrophage polarization in the liver, we detected macrophage polarization by cell markers and related cytokines. Importantly, M2 plays a crucial role in the prognosis of AHF, which results in altered levels of anti-inflammatory and pro­inflammatory factors. The mechanism by which M2 macrophages participate in the activation of infused MSCs remains unclear. In such a situation, the observed differential effects of M1 and M2

macrophages suggest that M2 polarization may provide a potential therapeutic application in AHF after MSC transplantation.

ARTICLE HIGHLIGHTSResearch backgroundRecent studies have demonstrated that macrophages promote stem cell activity via paracrine action. Macrophages can express multi-phenotype and multi-functional roles in the liver and are a major source of both pro-proliferative and anti-proliferative mediators in liver pathology. There is little information available on the role of macrophage polarization in rescuing acute hepatic failure by mesenchymal stem cells.

Research motivationDifferent macrophage phenotypes play various roles in tissue damage and maintenance. It is not clear whether M1 or M2 polarization contributes to the

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Figure 6 Flow cytometry analysis for serum levels of IFN-y and IL-4 in survival and death groups after mesenchymal stem cell treatment. Bar represents the mean ± SD (n = 6, bP < 0.001).

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Figure 7 mRNA expression of M1- and M2-related factors in survival and death groups after mesenchymal stem cells treatment. The mRNA expression of M1-related factors (TNF-α, INOS, and CD68) and M2-related factors (Arg-1, IL-10, and CD163) was detected in the total liver tissues of death group and survival group after MSC treatment. Bar represents the mean ± SD (n = 6, aP < 0.05, bP < 0.001).

ARTICLE HIGHLIGHTS

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therapeutic effects of mesenchymal stem cells (MSCs). Macrophages to M1 or M2 polarization can increase infused MSCs activity during MSC transplantation, and improve the clinical efficacy of MSCs in the treatment of acute hepatic failure.

Research objectivesTo investigate whether M1 or M2 polarization contributes to the therapeutic effects of MSCs.

Research methodsThe rats were divided into a survival group and a death group at 48 h after MSC treatment. The rats in the survival group were still in good physical condition at 48 h after MSC treatment. The rats in the death group were in poor physical condition or in the state of death before they died at 48 h after MSC treatment. The polarization of M1 and M2 was compared between the two groups. Macrophage polarization was analyzed by M1 markers [CD68, tumor necrosis factor alpha (TNF-a), interferon-γ (IFN-γ), and inducible nitric oxide synthase (INOS)] and M2 markers [CD163, interleukin (IL)-4, IL-10, and arginase-1 (Arg-1)].

Research resultsThe number of CD163+ macrophages and levels of IL-4, IL-10, and Arg-1 were significantly up-regulated in the survival group. In contrast, CD68+ macrophages and levels of TFN-γ, TNF-α, and INOS were significantly up-regulated in the death group. However, a specific signaling pathway between macrophage polarization, associated cytokines, and hepatocyte regeneration has not been examined to date.

Research conclusionsThis study demonstrates that expression of hepatic progenitor surface marker (EpCAM) is the key to improving the prognosis of AHF. We detected macrophage polarization by cell markers and related cytokines. M2 macrophages play a crucial role in the prognosis of AHF, which results in altered levels of anti-inflammatory and pro-inflammatory factors. The mechanism by which M2 macrophages participate in activation of infused MSCs remains unclear. The observed differential effects of M1 and M2 macrophages suggest that M2 polarization may provide a potential therapeutic application in AHF after MSC transplantation.

Research perspectivesM2 macrophages and their associated cytokines can contribute to AHF rescuing by MSCs. It is unclear whether M2 related cytokines originate from the liver or from the implanted MSCs. Further localization studies and relevant cell experiments are needed to confirm the results.

ACKNOWLEDGMENTSThe authors would like to thank Li­Li Wang for her assistance with experiments.

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P- Reviewer: Hashimoto N, Kawakubo K, Ramsay MA, Tanabe S S- Editor: Gong ZM L- Editor: Wang TQ E- Editor: Huang Y

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